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A single intravenous injection of anti-Thy-1 monoclonal antibody (mAb) 1-22-3 is known to cause reversible mesangial proliferative glomerulonephritis. However, mAb 1-22-3 injection followed by unilateral nephrectomy leads to progressive glomerulosclerosis and tubulointerstitial change with an irreversible course. To identify genes that play an important role in the irreversible progression of renal injury, we used microarray technology to identify differences in gene expression between these models. Rats were intravenously injected with mAb 1-22-3 1 week after unilateral nephrectomy (irreversible model) or a sham operation (reversible model), and rats were killed on days 4, 7, 14, 42, and 56 after the injection. complementary DNA probes prepared from kidney messenger RNAs were hybridized with oligonucleotide microarrays containing 4854 rat genes. The microarray identified 189 differentially expressed genes, having at least a two-fold difference in expression level between the two models, and they were classified into five clusters. One of the clusters consisted of genes whose expression was markedly upregulated in the irreversible model. This cluster included the genes encoding osteopontin, kidney injury molecule-1, and thymosin beta10. Increased expression of thymosin beta10 was localized mainly in macrophages in the fibrotic interstitium, and upregulation of thymosin beta10 expression was also observed in a unilateral ureteral obstruction model. The microarray analysis yielded information on the molecular mechanisms responsible for the difference in disease progression between the reversible and irreversible model of anti-Thy-1 nephritis. Thymosin beta10 may play an important role in the progression of kidney disease.

Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.BackgroundCyclooxygenase-2 (COX-2) inhibitors are used as analgesics in postmenopausal women, who develop edema and require a salt-restricted diet. This study was performed to determine the renal expression of COX-2 and on COX-2–dependent regulation of renal blood flow (RBF) in ovariectomized rats.MethodsSprague-Dawley rats were divided into 4 groups: sham-operated rats fed a normal-salt diet (Sh+NS) or a low-salt diet (Sh+LS), and bilaterally ovariectomized rats fed a normal-salt diet (Ox+NS) or a low-salt diet (Ox+LS) (N = 6 in each group). Estrogen replacement therapy was performed on other ovariectomized rats. A renal clearance study was performed in anesthetized animals.ResultsOvariectomy increased renal cortical COX-2 expression independently of dietary salt intake (Sh+NS

Cloning and characterization of a novel gene promoting ureteric bud branching in the metanephros. Background The ureteric buds and metanephric mesenchymal cells reciprocally induce each other's maturation during kidney development, and implicated transcription factors, secreted growth factors, and cell surface signaling peptides are critical regulators of renal branching morphogenesis. Protein kinase C (PKC) is a key enzyme in the signal transduction mechanisms in various biologic processes, including development, because it regulates growth and differentiation. Inhibition of PKC by the sphingolipid product ceramide interferes with nephron formation in the developing kidney, but the molecule that controls ureteric bud branching downstream of PKC is still unknown. Methods Differential display polymerase chain reaction (PCR) of metanephroi cultured with a PKC activator and inhibitor was performed. We also examined the role of a novel gene in kidney development with organ culture system. Results A novel gene encoding a 759bp mRNA was identified, and we named it metanephros-derived tubulogenic factor ( MTF )/ L47 . Inhibition of MTF with antisense oligonucleotide impaired ureteric bud branching by cultured metanephroi, and addition of recombinant MTF protein promoted ureteric bud branching in cultured metanephroi and increased cell proliferation. Conclusion We identified a novel molecule in developing kidney that is capable of modulating ureteric bud branching and kidney differentiation.

Hepatocyte growth factor regulates proteoglycan synthesis in interstitial fibroblasts.BackgroundHepatocyte growth factor (HGF) is a clinically important growth factor with therapeutic potential for the treatment of interstitial fibrosis and chronic renal failure. Proteoglycans are components of the renal interstitium, which have multiple actions, including growth regulation. In this study, we examined the effects of HGF on proteoglycan synthesis in interstitial fibroblasts, and the mechanisms of these effects.Methods and ResultsExpression and agonist-induced activation of the HGF receptor c-Met was detected in rat renal interstitial fibroblasts (NRK-49F) by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immune complex/immunoblot assay. Moreover, stimulation of the cells with HGF resulted in a marked increase (five- to tenfold) in phosphorylation of extracellular signal-related protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), but not of c-Jun NH2 terminal kinase (JNK). Treatment with HGF resulted in a time- and dose-dependent increase (P < 0.01) in both cell-associated and secreted proteoglycan synthesis to two- to fourfold of control levels. This effect was attenuated by the MAPK/ERK kinase (MEK) inhibitor PD98059 and the p38MAPK inhibitor SB203580. Ion-exchange chromatography suggested that chondroitin sulfate/dermatan sulfate proteoglycans were up-regulated after HGF treatment. Northern blot, RT-PCR, Western blot, and promoter activity assays revealed that HGF caused a significant increase in decorin mRNA and protein, as well as in biglycan mRNA, protein, and promoter activity, suggesting transcriptional control of gene expression. Since the effects of biglycan on fibroblast proliferation are still unclear, the effects of biglycan were examined by thymidine assay, and biglycan was found to attenuate transforming growth factor-β (TGF-β)–induced changes in cell proliferation.ConclusionThese results suggest that HGF causes an increase in the small leucine-rich proteoglycans biglycan and decorin by ERK1/2- and p38MAPK-mediated pathways in fibroblasts. These findings may be relevant for understanding potential mechanisms by which HGF can exert TGF-β inhibitory actions in the kidney.

Gene transfer of truncated IκBα prevents tubulointerstitial injury. Background Severe proteinuria not only indicates the presence of progressive glomerular disease, but also causes tubular epithelial cells to produce inflammatory mediators leading to tubulointerstitial (TI) injury. We investigated the role of nuclear factor-κB (NF-κB) in tubular epithelial cells in the development of proteinuria-induced TI injury. Methods To specifically inhibit NF-κB activation, a recombinant adenovirus vector expressing a truncated form of IκBα (AdexIκBΔN) was injected into renal arteries of protein-overloaded rats, a model of TI injury characterized by infiltration of mononuclear cells and fibrosis. Results Activation of NF-κB in the renal cortex, observed in protein-overloaded rats treated with a control vector, recombinant lacZ adenovirus, was prevented in AdexIκBΔN-injected rats. Microscopic examination revealed AdexIκBΔN treatment to markedly attenuate proteinuria-induced TI injury. Increased immunostaining of vascular cell adhesion molecule-1, transforming growth factor-β, and fibronectin in TI lesions also was suppressed by AdexIκBΔN injection. Conclusions These findings provide evidence of the critical role of NF-κB activation in TI injury and suggest the therapeutic potential of adenovirus-mediated IκBΔN gene transfer into the kidney as a means of interrupting the process of TI damage.

Glucocorticoid regulation of proteoglycan synthesis in mesangial cells.BackgroundProteoglycans are integral components of the mesangial matrix and glomerular permeability barrier. Recent studies have shown that changes in glomerular proteoglycan expression may play a major role in the pathogenesis of renal disease. Steroid hormones are used as first-choice therapy for the treatment of glomerular diseases, however, the effects of glucocorticoids on expression of glomerular proteoglycans are unknown.MethodsThis study examined the effects of in vitro and in vivo administration of dexamethasone on proteoglycan synthesis and gene expression of proteoglycan core proteins using rat (RMC) and human (HMC) mesangial cells.ResultsTreatment of cultured RMC with dexamethasone resulted in a dose- and time-dependent decrease (P < 0.05) in both cell-associated and secreted proteoglycan synthesis to approximately 50% of control levels. This effect was inhibited by the glucocorticoid antagonist mifepristone, and mimicked by prednisolone or corticosterone treatment. Separation of proteoglycans by ion-exchange and gel permeation chromatography suggested that chondrotin sulfate/dermatan sulfate proteoglycans were down-regulated after steroid treatment. Northern blot analysis, RT-PCR, Western blot, and promoter activity assays revealed that dexamethasone caused a significant decrease in decorin mRNA (to 61 ± 8% of controls), whereas biglycan expression and promoter activity were increased after steroid treatment. A similar trend was found in glomeruli isolated from rats treated in vivo with dexamethasone.ConclusionsThese results demonstrate that treatment of mesangial cells with steroids results in a decrease in total proteoglycan synthesis, as well as subtype-specific changes in proteoglycan core protein gene expression by transcriptional control, furthering our understanding of the effects of steroid treatment on the renal glomeruli.

In vivo visualization of angiotensin II- and tubuloglomerular feedback-mediated renal vasoconstriction.BackgroundA noninvasive technique to monitor renal microcirculation would be a useful tool for investigation of renal disease and the effects of drugs on the renal system. We have developed a novel, less invasive technique to visualize renal microcirculation in vivo using an intravital tapered-tip (1mm ø) lens-probe (pencil lens-probe) videomicroscopy, which only requires insertion of the probe into superficial renal cortex in situ.MethodsTo assess validity of this technique, the effects of angiotensin II (Ang II) and intrarenal sodium chloride loading (activator of tubuloglomerular mechanism) were examined. The renal microvasculature was successfully visualized and monitored.ResultsAdministration of Ang II (1, 3, 10 and 30 ng/kg/min) produced a dose-dependent constriction of afferent and efferent arterioles in similar degrees; at 30 ng/kg/min, Ang II elicited 52 ± 3 (N = 9) and 53 ± 3% decreases in diameter (N = 9), respectively. The Ang II-induced arteriolar constriction was completely prevented by losartan, an Ang II type 1 (AT1) antagonist. The intrarenal hypertonic saline administration elicited transient increments (from 98 ± 8 to 122 ± 7 mL/min, N = 6, P < 0.05), followed by a marked reduction in renal blood flow (RBF; 78 ± 7 mL/min, P < 0.05). This response was accompanied by prominent constriction of afferent (from 15.0 ± 1.1 to 8.5 ± 1.1 μm, N = 6, P < 0.05), but not efferent (from 14.3 ± 1.2 to 13.8 ± 1.0 μm, N = 3) arterioles. Furthermore, this response was completely inhibited by furosemide, a tubuloglomerular feedback inhibitor.ConclusionThe intravital pencil lens-probe videomicroscopy can be a powerful tool for in vivo observation of renal microcirculation, with intact renal microvascular responses to two important renal homeostatic mechanisms, angiotensin II and tubuloglomerular feedback.

Overexpression of truncated IkBα potentiates TNF-α-induced apoptosis in mesangial cells. Background Dysregulation of apoptosis is one of the likely underlying mechanisms of mesangial proliferative glomerulonephritis (GN), a disease in which proinflammatory cytokines exhibit a wide range of biological activities. Among them, tumor necrosis factor-α (TNF-α) induces two conflicting pathways, one leading to activation of the nuclear factor-kappa B (NF-kB), and the other leading to caspase-mediated apoptosis. We investigated whether or not specific inhibition of NF-kB affects TNF-α-induced apoptosis in rat mesangial cells (MCs). Methods To specifically inhibit NF-κ;B activation, we constructed a recombinant adenovirus vector expressing a truncated form of I kappa Bα (AdexIκBΔN) that lacks the phosphorylation sites essential for the activation of NF-κ;B. Electrophoretic mobility shift assay was performed to evaluate NF-kB activity. Nuclear morphology was observed by staining with Hoechst-33258. DNA fragmentation was detected using an ELISA kit with an antihistone antibody. To investigate the regulation of apoptosis, we measured caspase-3 and caspase-8 activity by ELISA, and examined the Bcl-2 and Bax protein level by Western blot. Results TNF-α-induced NF-κ;B activation was blocked by overexpression of IκBΔN. Overexpression of Iκ;BΔN potentiated TNF-α-induced apoptosis compared to mock transfection, and the potentiation was abolished by treatment with a caspase-3 inhibitor, Z-DEVD-FMK. Overexpression of IκBΔN augmented TNF-α-induced caspase-3 and caspase-8 activity, but did not affect Bcl-2 or Bax protein expression. Conclusion Overexpression of IκBΔN potentiates TNF-α-induced apoptosis and augments caspase-8 and caspase-3 activity in rat MCs without changing Bcl-2 or Bax protein expression. These results suggest the potential usefulness of AdexIκ;BΔN to induce apoptosis in MCs under inflammatory conditions.

Two novel 1α-hydroxylase mutations in French-Canadians with vitamin D dependency rickets type I. Background Vitamin D dependency rickets type I (VDDR-I) is an autosomal recessive disorder in which 25-hydroxyvitamin D 1α-hydroxylase (1α-hydroxylase) activity in renal proximal tubules is deficient. VDDR-I is recognized throughout the world, but occurs more frequently in a subset of the French-Canadian population. We and others have recently cloned the human 1α-hydroxylase cDNA and gene, making it possible to screen for mutations. The first VDDR-I mutations were reported in one American and four Japanese patients. In this study, we screened for 1α-hydroxylase mutations in French-Canadian patients with VDDR-I. Methods The nine exons of the 1α-hydroxylase gene were amplified by polymerase chain reaction (PCR) from genomic DNA of four unrelated French-Canadian patients with VDDR-I and their parents, and sequenced. Results Three of the patients were homozygous for a single base-pair deletion (G) at position 262 in the cDNA that lies in exon 2, and causes a premature termination codon upstream from the putative ferredoxin- and heme-binding domains. The fourth patient was homozygous for a 7-bp insertion (CCCCCCA) at position 1323 of the cDNA that lies in exon 8, and causes a premature termination upstream from the putative heme-binding domain. In each family, obligate carriers have one copy of the mutant allele. These mutations, which could be detected by PCR-restriction fragment length polymorphism and polyacrylamide gel electrophoresis of the PCR products, were not found in 25 normal French-Canadians. Conclusion We describe two novel 1α-hydroxylase mutations that are consistent with loss of function in four French-Canadian patients with VDDR-I and suggest that the 1α-hydroxylase mutations arise from more than one founder in this population.

Interleukin (IL)-1 and IL-4 synergistically stimulate NF-IL6 activity and IL-6 production in human mesangial cells. Background While interleukin (IL)-4 inhibits pro-inflammatory cytokine expression by human monocytes, we have observed that it potentiates IL-6 production by IL-1-activated human mesangial cells (MC). To study the mechanism of this cell-type specific interaction between IL-1 and IL-4 in MC, we examined the effect of both cytokines on the activities of nuclear factor κB (NF-κB) and nuclear factor IL-6 (NF-IL6), transcription factors that are essential for IL-6 gene expression. Methods We evaluated IL-6 synthesis, mRNA expression, and mRNA stability by ELISA, Northern analysis, and the actinomycin D method, respectively. Activities of NF-κB and NF-IL6 were analyzed by gel shift assay. Results IL-4 augmented the IL-1 stimulated IL-6 mRNA levels by about threefold without altering mRNA stability. IL-1 treatment rapidly induced the binding activity of NF-κB. In contrast, IL-4 did not affect basal and IL-1-induced NF-κB activities. Both IL-1 and IL-4 stimulated NF-IL6 activity as early as 30minutes after treatment. When MC were treated with both cytokines together, marked activation of NF-IL6 was observed at five hours. Conclusions These results suggest that simultaneous activation of NF- κB and NF-IL6 is essential for IL-6 gene expression and that IL-1 and IL-4 cooperatively stimulate MC IL-6 production through their synergistic activation of NF-IL6.

Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney. Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) serve as intracellular calcium release channels involved in signal transduction of various hormones in the kidney. Molecular cloning studies have shown that there are three types of IP3R, designated type 1, type 2, and type 3. To characterize their localizations in the rat kidney, we employed immunohistochemical studies using type-specific monoclonal antibodies that were raised against the 15 C-terminal amino acids of each type of IP3R. Type 1 was detected in glomerular mesangial cells and vascular smooth muscle cells. Type 2 was expressed exclusively in intercalated cells of collecting ducts from the cortex to the inner medulla. Type 3 was expressed in vascular smooth muscle cells, glomerular mesangial cells, and some cells of cortical collecting ducts, probably principal cells. As to the subcellular distribution, type 1 and type 2 showed a homogenous distribution in the cytoplasm, whereas type 3 was present mainly in the basolateral portion of the cytoplasm. These results indicate that IP3R isoforms were expressed in a cell-specific manner. The heterogeneous subcellular localizations among the IP3R types suggests compartmentalization of distinct IP3-sensitive Ca2+ pools.

Effects of insulin on rat renal microvessels: Studies in the isolated perfused hydronephrotic kidney. Although insulin is demonstrated to decrease vascular tone, the role of insulin in renal microcirculation has not been fully determined. In the present study, the effect of insulin on renal microvascular tone was assessed using the isolated perfused hydronephrotic rat kidney. Insulin (300 µ U/ml) had no effect on the basal renal microvessel diameter. In addition, insulin did not alter myogenic (that is, pressure-induced) constriction of preglomerular microvessels, with similar magnitude of constriction observed in response to elevated renal perfusion pressure from 80 to 180mm Hg (interlobular artery, -23 ± 3% vs. -19 ± 4%; afferent arteriole, -22 ± 3% vs. -21 ± 4%, for control and insulin, respectively). In striking contrast, insulin dose-dependently reversed the norepinephrine (NE)-induced tone of interlobular arteries, afferent arterioles, and efferent arterioles, with 94 ± 9%, 104 ± 6%, and 86 ± 10% reversal at 300 µ U/ml, respectively. These vasodilator actions were markedly inhibited by N-Arg; in the presence of N-Arg, insulin (300 µ U/ml) exerted only a modest dilator action on interlobular arteries (24 ± 9% reversal), afferent arterioles (23 ± 10% reversal), and efferent arterioles (14 ± 9% reversal). A similar renal microvascular responsiveness to insulin was also observed during angiotensin II (Ang II)-induced constriction. In conclusion, the ability of insulin to dilate the renal microvasculature differs, with marked inhibitory action during NE/Ang II-induced constriction and almost no inhibition during myogenic constriction. Furthermore, the present study suggests that the insulin-induced renal vasodilation is mediated by nitric oxide.

Role of adenosine in the renal responses to contrast medium. Despite the development of non-ionic radiographic contrast media (CM), CM-induced nephropathy is a clinically important problem in patients with pre-existing renal insufficiency. We examined the effects of non-ionic CM (iohexol) on renal function in conscious dogs with and without renal insufficiency, and evaluated the effects of a non-selective (theophylline), an A1 selective (KW-3902), and an A2 selective adenosine antagonist (KF17837) on the renal responses to CM. In sham-operated group, iohexol (2ml/kg/min for 3min) increased effective renal plasma flow (ERPF) and glomerular filtration rate (GFR), whereas in renal insufficiency group (with subtotal nephrectomy), following transient increases in ERPF and GFR, CM markedly decreased ERPF (-46.5 ± 6.7%) and GFR (-51.2 ± 7.1%). In sham-operated group, theophylline and KF17837 markedly attenuated CM-induced increases in ERPF and GFR, while KW-3902 had no effects on CM-induced increases in ERPF or GFR. In renal insufficiency group, initial increases in ERPF and GFR were blunted by theophylline and KF17837. In contrast, the subsequent decreases in ERPF and GFR were attenuated by theophylline (%ΔERPF, -12.2 ± 3.2% vs. -46.6 ± 6.7%, P < 0.01; %ΔGFR, 4.3 ± 2.5% vs. -51.0 ± 7.1%, P < 0.01), and were completely prevented by KW-3902 (%ΔERPF, 10.8 ± 2.9%; %ΔGFR, 23.8 ± 4.4%), whereas KF17837 aggravated ERPF (-73.3 ± 5.3%) and GFR (-78.4 ± 5.3%). These data indicate that in normal renal function, iohexol elicits renal vasodilation by activating mainly the adenosine A2 receptors. In contrast, in impaired renal function, CM induces both A2 and A1 activation; the former is associated with the initial renal vasodilation, while the latter is responsible for the sustained aggravation of renal hemodynamics.

Severe proteinuria not only indicates the presence of progressive glomerular disease, but also causes tubular epithelial cells to produce inflammatory mediators leading to tubulointerstitial (TI) injury. We investigated the role of nuclear factor-kappaB (NF-kappaB) in tubular epithelial cells in the development of proteinuria-induced TI injury.To specifically inhibit NF-kappaB activation, a recombinant adenovirus vector expressing a truncated form of IkappaBalpha (AdexIkappaBDeltaN) was injected into renal arteries of protein-overloaded rats, a model of TI injury characterized by infiltration of mononuclear cells and fibrosis.Activation of NF-kappaB in the renal cortex, observed in protein-overloaded rats treated with a control vector, recombinant lacZ adenovirus, was prevented in AdexIkappaBDeltaN-injected rats. Microscopic examination revealed AdexIkappaBDeltaN treatment to markedly attenuate proteinuria-induced TI injury. Increased immunostaining of vascular cell adhesion molecule-1, transforming growth factor-beta, and fibronectin in TI lesions also was suppressed by AdexIkappaBDeltaN injection.These findings provide evidence of the critical role of NF-kappaB activation in TI injury and suggest the therapeutic potential of adenovirus-mediated IkappaBDeltaN gene transfer into the kidney as a means of interrupting the process of TI damage.

Role of protein kinase C in angiotensin II-induced constriction of renal microvessels Background Although angiotensin II (Ang II) exerts its action through multiple vasomotor mechanisms, the contribution of phosphoinositol hydrolysis products to Ang II-induced renal vasoconstriction remains undetermined. Methods The role of protein kinase C (PKC) in Ang II-induced afferent (AFF) and efferent (EFF) arteriolar constriction was examined using the isolated perfused hydronephrotic rat kidney. Results Ang II (0.3 nmol/L)-induced EFF constriction was refractory to inhibition of voltage-dependent calcium channels by pranidipine (1 μmol/L, 19 ± 2% reversal) but was completely reversed by a PKC inhibitor, chelerythrine (1 μmol/L, 96 ± 2% reversal). Furthermore, direct PKC activation by phorbol myristate acetate (PMA; 1 μmol/L) caused prominent EFF constriction, and this constriction was inhibited by manganese and free calcium medium. In contrast, Ang II-induced AFF constriction was completely abolished by pranidipine (98 ± 4% reversal) and was partially inhibited by chelerythrine (55 ± 3% reversal). Although PMA elicited marked AFF constriction, this constriction was insensitive to the calcium antagonist, but was totally inhibited by manganese or free calcium medium. Conclusions PKC plays an obligatory role in Ang II-induced EFF constriction that requires extracellular calcium entry through nonselective cation channels. In contrast, in concert with our recent findings demonstrating a complete dilation by thapsigargin, Ang II-induced AFF constriction is mainly mediated by inositol trisphosphate (IP 3 ) and voltage-dependent calcium channel pathways, but could not be attributed to the PKC-activated calcium entry pathway (for example, nonselective cation channels). Rather, Ang II-stimulated PKC may cross-talk to the IP 3 /voltage-dependent calcium channel pathway and could modulate the vasoconstrictor mechanism of the AFF. Thus, the role of PKC during Ang II stimulation differs in AFF and EFF, which may constitute segmental heterogeneity in the renal microvasculature.

Vascular endothelial growth factor activates MAP kinase and enhances collagen synthesis in human mesangial cells. Background Vascular endothelial growth factor (VEGF) is an endothelial mitogen that is constitutively expressed in normal human glomeruli, but its role in the kidney is still unclear. In this study, we examined the effects of VEGF on human mesangial cells (HMCs). Methods and Results Reverse transcription-polymerase chain reaction analysis demonstrated the presence of VEGF receptor mRNA (flt-1 and KDR) in HMCs. The treatment of HMCs with VEGF did not cause a change in 3 H-thymidine incorporation or cell numbers. In contrast, VEGF caused a dose- and time-dependent increase in collagen synthesis, with threefold to fivefold increases in both cell-associated and secreted collagen synthesis seen after treatment with 200 ng/ml VEGF. The effects of VEGF were attenuated by treatment of HMCs with the tyrosine kinase inhibitor herbimycin A or the MEK inhibitor PD 98059, but not with the protein kinase C (PKC) inhibitor chelerythrine. VEGF treatment also caused a marked increase in p42/p44 mitogen-activated protein kinase (MAPK) activity, but had no significant effect on HMC superoxide production. Finally, an increase in collagen synthesis was also seen in rat mesangial cells treated with VEGF. Conclusions These results suggest that VEGF is not a mitogenic signal in HMCs, but may be involved in the regulation of the mesangial matrix in humans by a MAPK-dependent mechanism.

Role of chloride channels in afferent arteriolar constriction. The effects of IAA-94, a chloride channel blocker, and/or low chloride perfusate on afferent arteriolar (AA) constriction by angiotensin II (Ang II), norepinephrine (NE) and increasing pressure (80 to 160mmHg) were assessed, using isolated perfused hydronephrotic kidneys. In the first series of experiments, Ang II (0.3nM) constricted AAs by 33 ± 3% (N = 5, P < 0.01). Subsequent addition of diltiazem (10 µM) restored the decrements in the AA diameters. In the presence of diltiazem (10 µM), increasing pressure did not constrict AAs. In the second series of experiments, elevation of pressure constricted AAs by 20 ± 2% (N = 7, P < 0.01). Subsequent addition of IAA-94 (30 µM) failed to alter the basal AA diameter and myogenic responsiveness. However, Ang II-induced AA constriction was abolished by IAA-94. In the third series of experiments, decreasing extracellular chloride exaggerated AA constriction by 0.1nM of Ang II (from 13 ± 2 to 20 ± 3%, N = 6, P < 0.05). Similarly, low chloride perfusate enhanced NE (0.1 µM)-induced AA constriction (from 14 ± 2 to 19 ± 2%, N = 6, P < 0.05). In contrast, myogenic responsiveness was not influenced by reducing chloride concentrations. The present data provide evidence that both Ang II and NE induce AA constriction by opening chloride channels and subsequent activation of voltage-dependent calcium channels, and suggest that the myogenic response is mediated by activating voltage-dependent calcium channels independently of chloride channels.

Angiotensin II type 1 receptor gene abnormality in a patient with Bartter's syndrome. Administration of a selective angiotensin I type 1 receptor (AT 1 ) antagonist in animals not only nullifies the vasopressor action of angiotensin II, but also induces chloriduria and kaliuria, juxtoglomerular apparatus (JGA) hypertrophy and hyperreninemia, features characteristic of human Bartter's syndrome. We, therefore, explored the possibility that Bartter's syndrome may involve an AT 1 abnormality. Using a pair of AT 1 -specific oligonucleotide primers and two different DNA polymerases ( Taq and Pfu ), we amplified the ˜1kb AT 1 coding region of genomic DNA isolated from leukocytes of five patients with Bartter's syndrome by PCR and analyzed the sequence of the product. While the sequence of all clones from four patients were identical to that already reported for the normal human AT 1 DNA sequence, 50% of the clones from one patient with Bartter's syndrome were found to have A → G transition at nucleotide 931 which causes an amino acid substitution (arg → gly) on the carboxy-terminal cytosolic tail of AT 1 . This mutation was not found in DNA from 50 normal controls which were screened by restriction enzyme digestion pattern of the PCR products of this region. As PCR-amplified AT 1 DNA clones from four other individuals with Bartter's syndrome did not display any abnormality in the coding region, the possibility exists that Bartter's syndrome consists of multiple disease entities, where an AT 1 gene abnormality represents a specific subgroup of the syndrome and/or some abnormality includes mutations outside of the coding region.

Transient receptor potential channels in rat renal microcirculation: Actions of angiotensin II. Background This study assessed the calcium-activating mechanisms mediating glomerular arteriolar constriction by angiotensin II (Ang II). Methods Immunohistochemical and physiological studies were carried out, using antibody against transient receptor potential (TRP)-1 and an isolated perfused kidney model. Results Immunohistochemical experiments demonstrated that TRP-1 proteins were transcribed on both afferent and efferent arteriolar myocytes. In the first series of physiological experiments, Ang II (0.3 nmol/L) considerably constricted afferent (20.2 ± 0.9 to 14.9 ± 0.7 μm) and efferent arterioles (18.4 ± 0.7 to 14.0 ± 0.7 μm). The addition of nifedipine (1 μmol/L) restored decrements in afferent (to 20.0 ± 0.8 μm) but not efferent arteriolar diameters. Further administration of SKF-96365 (100 μmol/L), a TRP channel blocker, reversed efferent arteriolar constriction (to 16.2 ± 0.8 μmol/L). In the second group, although 2-aminoethoxydiphenyl borate (100 μmol/L), an inhibitor of inositol trisphosphate-induced calcium release (IP3CR), did not alter glomerular arteriolar diameters, it prevented Ang II-induced afferent arteriolar constriction and attenuated efferent arteriolar constriction (18.8 ± 0.8 to 16.9 ±μm). Subsequent removal of extracellular calcium abolished residual efferent arteriolar constriction (to 19.1 ± 0.8 μm). Conclusions Our data provide evidence that Ang II elicits IP3CR, possibly inducing a cellular response that activates voltage-dependent calcium channels on afferent arterioles. The present results suggest that Ang II-induced efferent arteriolar constriction involves IP3CR and calcium influx sensitive to SKF-96365.

Identification of 25-hydroxyvitamin D 3 1α-hydroxylase gene expression in macrophages. Background The 25-hydroxyvitamin D 3 1α-hydroxylase (1α-hydroxylase) is almost exclusively expressed in the kidney. However, 1α-hydroxylase activities have been observed in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage. In sarcoidosis, macrophage 1α-hydroxylase causes overproduction of 1,25-(OH) 2 D 3 , resulting in hypercalcemia. In this study, we investigated the regulation of macrophage 1α-hydroxylase at a molecular level. Methods We used the human monocytic cell line THP-1, which can be differentiated into macrophage-like cells by treatment with phorbol ester. The expression of 1α-hydroxylase in THP-1 cells was examined by Northern blotting and immunoblotting using an antibody raised against a synthetic peptide corresponding to the 14 C-terminal amino acids of 1α-hydroxylase. We investigated the regulation of 1α-hydroxylase mRNA expression by RNase protection assay. Results Northern blot and immunoblot analyses confirmed the expression of 1α-hydroxylase in THP-1 cells at the mRNA and protein levels. Although parathyroid hormone and calcitonin, known stimulators of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA, 8-Br-cAMP (5 × 10 -4 mol/L) increased the expression of 1α-hydroxylase mRNA in THP-1 cells (198 ± 9%). 1,25-(OH) 2 D 3 , known as a suppressor of renal 1α-hydroxylase, did not affect the expression of 1α-hydroxylase mRNA. By contrast, 1,25-(OH) 2 D 3 markedly increased the expression of 25-hydroxyvitamin D 3 24-hydroxylase mRNA. Interferon-γ (2000 IU/mL) increased the expression of 1α-hydroxylase mRNA in differentiated THP-1 cells (922 ± 25%). Conclusions The present results suggest that 1α-hydroxylase activity in macrophages is mediated by the same enzyme as in kidney. Interferon-γ treatment increases macrophage 1α-hydroxylase levels via directly increasing gene expression of this enzyme.

Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats. Background Although Rho-kinase is reported to play an important role in vascular injury, the contribution of Rho-kinase to the progression of renal injury remains unestablished. Methods We examined the effect of fasudil, a Rho-kinase inhibitor, on the progression of renal injury in subtotally nephrectomized spontaneously hypertensive rats (SHR). Rats were randomly assigned to three groups: sham-operated SHR; salt-loaded subtotally nephrectomized rats (SHR-subtotal nephrectomy); SHR-subtotal nephrectomy given fasudil for 6weeks (SHR-subtotal nephrectomy + fasudil; 3mg/kg/day). Renal morphologic and molecular analysis as well as urinary protein excretion was evaluated. Results In SHR-subtotal nephrectomy treated with fasudil, systolic blood pressure was not significantly different from that in SHR-subtotal nephrectomy without fasudil (208 ± 8mm Hg vs. 217 ± 14mm Hg). Urinary protein excretion was markedly increased in SHR-subtotal nephrectomy (124 ± 16mg/day), but this increase was significantly suppressed by fasudil (79 ± 12mg/day). Renal histologic examination revealed that fasudil improved glomerular and tubulointerstitial injury scores with parallel amelioration of proliferating cell nuclear antigen-positive and ED-1–positive cell infiltration. Furthermore, Western blot analyses showed that both expression and activity of Rho-kinase were enhanced in SHR-subtotal nephrectomy, compared with those in SHR without nephrectomy, and fasudil suppressed Rho-kinase activity. Finally, fasudil up-regulated the expression of p27 kip1 , a cyclin-dependent kinase inhibitor, and increased the p27 kip1 immunopositive cells in both glomeruli and tubulointerstitium with the use of immunohistochemistry. Conclusion Rho-kinase pathway is involved in the pathogenesis of renal injury. Furthermore, the inhibition of Rho-kinase may constitute a therapeutic strategy for the treatment of renal injury in part through the p27 kip1 up-regulation and the subsequent inhibition of cell proliferation and macrophage recruitment.